It is well known that the genetic information of all cells is stored in deoxyribonucleic acid (DNA) in the chromosomal material of organisms. The units of genetic function, i.e., the locus on the chromosome related to a specific hereditary trait, is called a gene.
Recombinant DNA technology involves the transfer of genetic material (genes) from one organism into a second organism and the propagation of these combined materials in bacterial and animal cells. The cell into which the recombinant genetic material is inserted is designated the host.
In the 1950's it was discovered that bacterial cells contain circular extra chromosomal DNA molecules, called plasmids, in addition to the main DNA molecule. The plasmids contain a series of genes, linked together in the form of a circle. These plasmids are small, easy to handle in the laboratory and enter other bacteria with ease. Plasmids represent a class of DNA molecules which accept DNA fragments and are referred to as the vector component of the hostvector system. Subsequently it was discovered that bacterial cells contain restriction enzymes that act as "chemical scalpels" to split DNA molecules into specific fragments which usually contain from less than 1 to 10 genes each. These specific fragments are the genetic material that will be inserted into the vector. The combined DNA fragment and vector are referred to as recombinant DNA. Restriction enzymes cleave viral DNA in the same manner as they cleave the plasmid DNA. Viruses represent another class of vectors.
Using recombinant DNA technology, genetic exchange between bacteria can be accomplished as follows. Plasmid or viral DNA (vector) is first isolated. Plasmid DNA is then linearized by cleaving or breaking the molecule at a single site, either by the use of restriction enzymes or other means. The DNA to be inserted (for example chromosomal) into the vector is also cleaved with restriction enzymes or other well known techniques designed to break the DNA into fragments. A fragment for the desired genetic characteristic is then inserted into the "broken" plasmid (the vector DNA ring). By treatment with DNA ligase the ends are joined and a recombinant plasmid DNA molecule is formed. The recombinant plasmid DNA molecule contains the genes of the bacterial plasmid plus the new genes from the inserted fragment. This plasmid can be introduced into a bacterium host. The new genes are propagated and become a part of the genetic machinery of the bacterium. In order to be useful for recombinant DNA technology, the microorganism (host) must be capable of undergoing "transformation", i.e., itself be capable of incorporating DNA and yielding a viable microorganism capable of expressing the traits encoded by the newly inserted genes. In this way the microorganism (host) can incorporate other desirable generic characteristics from other organisms.
It has been clear from the beginning of experimentation in recombinant DNA technology that novel gene combinations may have a potential for biological hazard, in that novel microorganisms capable of releasing products harmful for man, plants, or animals, may be produced. In order to prevent the spread of potentially harmful microorganisms, appropriate containment safeguards were investigated.
Containment of potentially biohazardous agents can be achieved in several ways. In 1978, the Direction of the NIH issued "Guidelines for Research Involving Recombinant DNA Molecules" (FR 43 60108) which set forth containment provisions. Physical containment was approached by using a set of microbiological standards which have been developed over a period of years for handling pathogenic organisms in research and clinical laboratories.
An equally important containment approach, because it contributes most significantly to limiting the spread of any potentially biohazardous agent, is the use of biological containment safeguards. Biological containment can be defined as the use of host cells and vectors with limited ability to survive outside of very special and fastidious conditions which can be maintained in the laboratory but are unlikely to be encountered by escaped organisms in natural environments.
The NIH Guidelines established levels of biological containment for host-vector systems (designated HV), dependent upon the microorganism and the DNA used. HVl is defined as a "host-vector system which provides a moderate level of containment."
In 1979, the NIH issued "Actions" (FR 44 71) under the NIH Guidelines which included criteria for consideration of B. subtilis for certification as a host in a HVl system. The FR Action stated that:
"Asporogenic mutant derivatives of B. subtilis can be accepted as the host component of an HVl system. These derivatives must not revert to sporeformers with a frequency greater than 10.sup.-7 ; data confirming this requirement must be presented to NIH for certification."
Emphasis was placed on eliminating formation of spores by the mutants because certain Bacillus species have developed a specialized mechanism for survival which involves the formation of spores. Spores are in a state of latent life with no metabolic activity and an increased resistance to the lethal effect of heat, drying, freezing, deleterious chemicals and radiation. In order to be susceptible to biological containment, Bacillus microorganisms cannot be capable of functioning as efficient sporeformers.
The present invention describes a new asporogenous mutant of B. subtilis RUB 830 designated as B. subtilis RUB 331, (ATCC 31578) and a process for using it in a host-vector system.
The literature on sporulation and the formation of mutants is extensive. Formation of mutants, including asporogenous mutants, is described in Bacteriological Reviews, Vol. 33:48-71 (1969) and Vol. 40:908-962 (1976). Also J. Bacteriol. 81:823-829 (1961) describes transformation of B. subtilis. These references do not disclose production of an asporogenous mutant which has the phenotype characteristics of the present invention.